Capillary Electrophoresis in DNA Analysis · in DNA Analysis NEAFS Workshop Mystic, CT September 29-30, 2004 Dr. John M. Butler ... • Review and Test Mixture of dye-labeled PCR

NEAFS CE-DNA Workshop (Butler and McCord) Sept 29-30, 2004

http://www.cstl.nist.gov/biotech/strbase/NISTpub.htm 1

Capillary Electrophoresis in DNA Analysis

NEAFS WorkshopMystic, CT

September 29-30, 2004Dr. John M. Butler

Dr. Bruce R. McCord

Data Interpretation

Outline for Workshop

• Introductions• STR Analysis• Introduction to CE and ABI 310• Data Interpretation• Additional Topics – Real-time PCR and miniSTRs• Higher Throughput Approaches• Troubleshooting the ABI 310 (Participant Roundtable)• Additional Topics – Y-STRs, validation, accuracy• Review and Test

Mixture of dye-labeled PCR products from

multiplex PCR reaction

Sample Separation

Sample DetectionCCD Panel (with virtual filters)

Argon ion LASER (488 nm)

ColorSeparationFluorescence

ABI Prism spectrograph

Capillary

Sample Injection

SizeSeparation

Processing with GeneScan/Genotyper software

Sample Interpretation

Butler, J.M. (2005) Forensic DNA Typing, 2nd Edition, Figure 13.8, © Elsevier Science/Academic Press

Steps in STR Typing

ABI 310

480 and 9600GeneAmp 9700

NIST Instruments Used for STR Typing

ABI 3100

FMBIO III Gel Imager System

PowerPlex 16 BIO

Penta E

D18S51

D21S11

TH01

D3S1358

Penta D

CSF1PO

D7S820

D13S317

D5S818

D16S539

TPOX

D8S1179

VWA

FGA

Amelogenin

Profiler Plus™

COfiler™

SGM Plus™

Green I

Profiler™

Blue

TH01

Amel D16S539D7S820

CSF1POTPOX

D3S1358

D16S539 D18S51D21S11

Amel

Amel

D3S1358

D3S1358

D18S51D21S11

D8S1179

D7S820

D13S317D5S818

D19S433 D2S1338

FGAvWA

vWA

FGA

TH01

D3S1358 vWA FGA

D7S820D5S818D13S317

TH01CSF1POTPOX

D8S1179

vWATH01 CSF1PO

TPOXAmel FGAD3S1358

Amel

PCR Product Size (bp)We work with all of the different commercial STR kits

We work with all of the different commercial STR kits



Identifiler™ kit multiplex STR result

AMELD3

TH01

TPOX

D2

D19

FGAD21D18

CSFD16

D7

D13D5 VWA

D8

PowerPlex® 16 kit multiplex STR result

AMEL

D3 TH01TPOX

Penta D

Penta E

FGA

D21 D18 CSF

D16

D7D13

D5

VWA

D8

Same DNA Sample with Two Commercial 16plex STR Typing Kits

NIST SRM 2391b Component 1

U.S. Population Study with Identifiler STR Kit

Identifiler™

J. Forensic Sci. 48(4):908-911

0.00000

0.10000

0.20000

0.30000

0.40000

0.50000

5 6 7 8 9 9.3 10 11

Allele

Freq

uenc

y

302 Caucasians 258 African Americans 140 HispanicsTH01302 Caucasians 258 African Americans 140 Hispanics

5 0.00166 0.003886 0.23179 0.12403 0.214297 0.19040 0.42054 0.278578 0.08444 0.19380 0.096439 0.11424 0.15116 0.15000

9.3 0.36755 0.10465 0.2464310 0.00828 0.00194 0.0142911 0.00166

TH01 Allele Frequencies

5 µL PCR protocol on ABI 3100Reagents cost only $3.53 per sample

700 U.S. samples examined

http://www.cstl.nist.gov/biotech/strbase/NISTpop.htm

Sample Interpretation Overview

• Data collection on analytical instrument (e.g., ABI 310 or ABI 3100)

• Peak identification and sizing

• Correlation of peak sizes to STR allele repeat number through comparison with previously or concurrently run allelic ladder

• Review of “called” alleles with editing where needed according to laboratory-specific interpretation guidelines

• Transfer of final reviewed allele calls from STR profile to table of genotypes

Done completely or with aid of computer program(s)

Data Collection

Peak Identification

Data Review by Analyst/Examiner

Color Separation

Peak Sizing

Comparison to Allelic Ladder

Confirmation of Results by Second Analyst/Examiner

Genotype Assignment to Alleles

GeneScansoftware

Genotypersoftware

Internal sizing standard

(e.g., GS500-ROX)

Matrix file

Allelic ladder sample

Steps in STR Genotyping ProcessData Collection

software

Expert Systems under Development

(e.g., True Allele)

Adapted from Butler (2001) Forensic DNA Typing, Figure 13.1

D3S1358 16,17vWA 17,18Amel X,Y (male)D8S1179 12,14

COfiler STR data

GeneScan view

Genotyperview

Allele call (repeat number)determined by comparison of peak size (bp) to allelic ladder allele peak sizes run under the same electrophoretic conditions

Peak height in relative fluorescence units (RFUs)

Three Possible Outcomes

• Match – Peaks between the compared STR profiles have the same genotypes and no unexplainable differences exist between the samples. Statistical evaluation of the significanceof the match is usually reported with the match report.

• Exclusion – The genotype comparison shows profile differences that can only be explained by the two samples originating from different sources.

• Inconclusive – The data does not support a conclusion as to whether the profiles match. This finding might be reported if two analysts remain in disagreement after review and discussion of the data and it is felt that insufficient information exists to support any conclusion.

Butler, J.M. (2001) Forensic DNA Typing, p. 202



Suspect 1

Suspect 2

Evidence

Match (Inclusion)

Exclusion (No match)

Crime Scene STR Profile Compared to Two Suspects Comparison of Allelic Ladder to Samples to Convert Size into Allele Repeat Number

difference = - 0.02 bp

difference = + 0.05 bp

+/- 0.5 bp bin defined around each allele

Precision from Run-to-Run on ABI 310

Size deviation of 70 samples and two allelic ladders from one injection of allelic ladder on a single ABI PRISM 310 Genetic Analyzer run

From Identifiler User’s Manual

Data to be Reviewed from Electropherograms

Identifiler kit ladder and sample data

Final Answer DesiredWhat is the D8S1179 genotype?Black labels = allele call (repeat #)

Blue labels = peak size (bp)Red labels = peak heights (RFUs)

Bleedthrough of allele from another dye color

Identifiler sample data

Stutter product219/4315 = 0.051

(5.1% of allele)

Black labels = allele call (repeat #)Blue labels = peak size (bp)Red labels = peak heights (RFUs)

Heterozygote peak imbalance3031/4315 = 0.702 = 70.2%

Stutter product260/3031 = 0.086

(8.6% of allele)

“off-ladder” (OL) allele?Often due to noisy baseline or bleedthrough from another dye

color (can sometimes be eliminated by raising the peak threshold—

like to 100 RFU in this case)

“-A” peak due to incomplete adenylation

Usually “-A” peaks will remain fairly constant in amount across allele range for a locus while stutter products increase in amount with larger alleles (higher number of repeat units)

444/

4315

= 0

.103

298/

3031

= 0

.098

9.8%10.3%

Important information from this sampleD8S1179 genotype = 11,14

SWGDAM STR Interpretation Guidelineshttp://www.fbi.gov/hq/lab/fsc/backissu/july2000/strig.htm

The interpretation of results in casework is a matter of professional judgment and expertise. Not every situation can or should be covered by a preset rule. It is important that the laboratory develops and implements written guidelines for interpretation of analytical results. This document provides a framework for the laboratory to develop short tandem repeat (STR) interpretation guidelines. The laboratory's interpretation guidelines should be based upon validation studies, data from the literature, instrumentation used, and/or casework experience.



1. Preliminary Evaluation of Data1.1. The laboratory should develop criteria to determine whether the results are of sufficient intensity/quality for interpretation purposes using methods appropriate for the detection platform. These criteria should be determined by evaluating data generated by the laboratory.

1.1.1. When quantitative results (e.g., peak amplitude) are used to evaluate STR profiles, the results should be examined to determine if they meet the laboratory's defined analytical and interpretational threshold(s).

1.1.1.1. The analytical threshold(s) is defined as the minimum and maximum intensity thresholds that are determined to assign alleles.

1.1.1.2. The interpretational threshold should be defined empirically.

1.1.2. When quantitative results are not used, the laboratory should establish criteria to interpret alleles based on visual inspection of gel images.

1.2. The laboratory should develop criteria to evaluate internal lane size standards and/or allelic ladders.

1.3. Controls are required to assess analytical procedures.

1.3.1. The laboratory should establish criteria for evaluation of the following controls, including but not limited to: reagent blank, amplification blank, and positive control.

1.3.2. The laboratory should develop criteria for the interpretation and documentation of results in the event that the controls do not perform as expected.

1.4. A laboratory using STR multiplexes that contain redundant loci should establish criteria regarding the concordance of such data.

http://www.fbi.gov/hq/lab/fsc/backissu/july2000/strig.htm

SWGDAM STR Interpretation Guidelines

2. Designation2.1. The laboratory should establish criteria to assign allele designations to appropriate peaks or bands.

2.1.1. Locus Designation: The laboratory should establish criteria to address locus assignment for alleles.

2.1.2. Allele Designation: The laboratory should designate alleles in accordance with Combined DNA Index System (CODIS) recommendations.

2.1.2.1. Whenever possible, allele designation should be based operationally on the number of repeat sequences contained within the allele and by comparison to an allelic ladder.

2.1.2.2. The designation of alleles containing an incomplete repeat motif (i.e., an off-ladder allele falling within the range spanned by the ladder alleles) should include the number of complete repeats and, separated by a decimal point, the number of base pairs in the incomplete repeat (e.g., FGA 18.2 allele).

2.1.2.3. If an allele falls above the largest or below the smallest allele of the allelic ladder, the allele should be designated as either greater than (>) or less than (<) the respective ladder allele, or when appropriate interpolation can be used.

2.2. Artifacts can occur and should be noted. These may include, but are not limited to, the following: pull-up, stutter, and nontemplate nucleotide addition. The laboratory should establish guidelines based on empirical data (obtained internally or externally) to address the interpretation of these and other artifacts.



3. Interpretation of Results3.1. The laboratory should define conditions in which the data would lead to the conclusion that the source of the DNA is either from a single person or more than one person. This may be accomplished by an examination of the number of alleles at each locus, peak height ratios, and/or band intensities.

3.1.1. Single Contributor: A sample may be considered to be from a single contributor when the observed number of alleles at each locus and the signal intensity ratios of alleles at a locus are consistent with a profile from a single contributor. All loci should be evaluated in making this determination.

3.1.2. Mixtures With Major/Minor Contributors: A sample may be considered to consist of a mixture of major and minor contributors if there is a distinct contrast in signal intensities among the alleles. The difference is evaluated on a case-by-case context. All loci should be evaluated in making this determination.

3.1.3. Mixtures With a Known Contributor(s): In some cases, when one of the contributors (e.g., the victim) is known, the genetic profile of the unknown contributor may be inferred. Depending on the profiles in the specific instance, this can be accomplished by subtracting the contribution of the known donor from the mixed profile.

3.1.4. Mixtures With Indistinguishable Contributors: When major or minor contributors cannot be distinguished because of similarity in signal intensities or the presence of shared or masked alleles, individuals may still be included or excluded as possible contributors.

3.2. The laboratory should have guidelines for interpretation of partial profiles (i.e., profiles with fewer loci than tested) that may arise from degraded or limited quantity DNA or from the presence of polymerase chain reaction (PCR) inhibitors.

3.3. The laboratory should establish guidelines to interpret profiles that exhibit potential stochastic effects (e.g., allele dropout and/or substantial imbalance of alleles).



4. Conclusions

4.1. The laboratory should prepare guidelines for formulating conclusions resulting from comparisons of single source samples and mixtures with known reference samples.

4.1.1. General categories of conclusions include, but are not limited to: inclusion or match, exclusion or nonmatch, inconclusive or uninterpretable, and no results.



5. Statistical Interpretation

5.1. The source of the population database(s) used should be documented. Relevant population(s) for which the frequency will be calculated should be identified.

5.2. The formulas used in calculating the frequency of a DNA profile should be defined for the following:

5.2.1. Heterozygote profiles

5.2.2. Homozygote profiles

5.2.3. Composite profiles (i.e., multiple locus profiles)

5.2.4. Minimum allele frequencies

5.2.5. Mixture calculations

5.2.6. Biological relationships, where appropriate

5.3. When used, criteria for the declaration of source attribution should be documented.



6. References/Suggested Readings

Committee on DNA Forensic Science, National Research Council. An Update: The Evaluation of Forensic DNA Evidence. National Academy Press, Washington, DC, 1996.

DNA Advisory Board. Quality assurance standards for convicted offender DNA databasinglaboratories (approved April 1999), Forensic Science Communications (July 2000) 2. Available at www.fbi.gov/programs/lab/fsc/backissu/july2000/codispre.htm

DNA Advisory Board. Quality assurance standards for forensic DNA testing laboratories (approved October 1998), Forensic Science Communications (July 2000) 2. Available at www.fbi.gov/programs/lab/fsc/backissu/july2000/codispre.htm

DNA Commission, ISFH. DNA recommendations: 1994 report concerning further recommendations regarding PCR-based polymorphisms in STR (short tandem repeat) systems, Forensic Science International (1994) 69:103–104.

Federal Bureau of Investigation. National DNA Index System (NDIS) Procedures Manual.U.S. Department of Justice, Washington, DC, February 1999 (revised).





GeneScan and Genotyper Review of STR Data

• GeneScan– Apply matrix, size standard, and analysis parameters– Remove the peak designation from the “250 bp” peak in the

GS500 ROX/LIZ internal size standard– Confirm that all alleles in the allelic ladder have been

designated as peaks

• Genotyper– Scan sizes of all peaks in the internal size standard to

confirm that they are correct (especially the 340 bp peak)– Run genotyping macro (Kazaam or Power)– Review electropherograms and edit data primarily through

removing labels on peaks determined not to be alleles– Create a table of final allele calls for export

GeneScan® Software

• Calls peaks (based on threshold values)• Separates colors with matrix file• Sizes peaks with internal size standard

Macintosh

2.1

3.1

3.1.2 (5-dye)

Windows NT

3.7 (5-dye)

3.7.1 (5-dye)

ABI manual is P/N 4303189

GeneScan Software Screen Views

Raw Data

Sample Information

Size Curve

Instrument Performance Indicators (Current, etc.)

Analyzed Data

From Applied Biosystems User Bulletin, ABI Prism GeneScan Analysis Software for the Windows NT Operating System

Flowchart of Steps Used by GeneScan Software to

Produce Analyzed STR Data

Baseline Subtraction

Peaks are filtered to produce normalized data

Analysis Parameters



GeneScan Analysis ParametersMacintosh version 3.1

NT version 3.7 NT version 3.7.1 DefaultNT version 3.7 Default

GeneScan Analysis Parameter Differences

GeneScan® Analysis version 3.7.1 enables analysis of sample files that, under certain circumstances, might have failed under v3.7. Several minor improvements were implemented to increase the robustness of the sizecaller and improve the baselining function to eliminate negative peak area.

GeneScan® Analysis version 3.7.1 enables analysis of sample files that, under certain circumstances, might have failed under v3.7. Several minor improvements were implemented to increase the robustness of the sizecaller and improve the baselining function to eliminate negative peak area.

Heavy

None

Light

Effect of Smoothing Options

Baseline noise is reduced with smoothing but so is peak height along with resolution between closely spaced peaks (e.g., TH01 9.3/10)

D21S11 allelic ladder from Profiler Plus kit Differences between Mac and NT versions of 310 Data Collection

J. Forensic Sci. 2004, 49(1):92-95

The NT version of GeneScan captures peak height information prior to smoothing

2,979 peaks evaluated

Be Careful when Limiting the Analysis Range

Do not put too close to peaks that are needed

Start point (e.g., 4000 data points)

There will be some variation from run to run in terms of scan numbers

Sample runs a little slower

Sample runs a little faster

Peaks Seem to Disappear from Electropherograms if the Analysis Start Position is too Late

Genotyper 3.7 view

Must go back to GeneScan software and reduce Analysis start position to capture the 75 bp peak…



Genotyper 3.7 view

Peak appears out of +/-0.5 bp sizing window due to loss of 75 bp peak from the GS500 ROX sizing standard

Appearance of “poor precision” may be due to failure to include a peak in the internal size standard for that sample

Be careful about the stop point for Be careful about the stop point for data collection and analysisdata collection and analysis

Approx. 1 minute difference in these runsApprox. 1 minute difference in these runs

If a stop point of 25 minutes was used, the 400 bp peak would not be collected or analyzed…

There will be some variation from run to run in terms of scan numbers

Data from Debbie Hobson (2001) FBI Laboratory

Internal Size Standards and Sizing Algorithms

DNA fragment peaks in sample

DNA Size

Data Point

147.32 bp147.32 bp

165.05 bp165.05 bp

100

139150

160

200

250

DNA fragment peaks are sized based on the sizing curve produced from the points on the internal size standard

Process of Sizing DNA Fragments Using an Internal Standard

Butler, J.M. (2001) Forensic DNA Typing, Figure 13.2(b), ©Academic Press

Internal Sizing StandardsGS500 ROX (Applied Biosystems)

LTI 50-500 ROX (Life Technologies-no longer available)

ILS600 CXR (Promega)

GeneFlo 625 ROX (CHIMERx-no longer available?)

“250 peak” not included in calculations



Local Southern Sizing Algorithm• Local Southern is commonly used but may not be the best in all

situations• Local Southern involves using 2 peak above and 2 peaks below

an unknown peak from the internal size standard to make a calculated DNA size

Size of STR Allele

X1 X2 X1 + X2

2=

Internal size standard

STR Allele

Elder, J. K. and Southern, E. M. (1983) Measurement of DNA length by gel electrophoresis II: Comparison of methods for relating mobility to fragment length, Analytical Biochemistry 128:227-231.

Normally the 250 bp peak is not included because it does not always migrate uniformly compared to the other peaks in the size standard

Global Southern vs Local Southern MethodsGlobal Southern Sizing

There is value in Global Southern Sizing for STRs when temperature variations within a run exist:– Hartzell, B., Graham, K., McCord,B. (2003) Forensic Sci. Int.

133:228-234– Klein et al. (2003) Forensic Sci. Comm. 5(1)

• Global Southern Method: Generates best-fit curve from all matched fragments in the size standard

• Local Southern Method: Generates best-fit curve from only nearby internal lane standard data points (usually two peaks on either side of the unknown)

Abstract

Early studies have established the Local Southern algorithm as a precise tool for sizing DNA fragments. As a result, the Local Southern algorithm of the PE Applied Biosystems' software, GeneScan® Analysis (PE Applied Biosystems, Foster City, California), is the manufacturer's recommended method for sizing short tandem repeats (STRs). However, this recommendation is made with the warning that size estimates may be imprecise if any of the standard fragments run anomalously. Specifically, the GeneScan®-500 (GS-500) internal standard fragments of 250 and 340 bases in length run anomalously under non-optimal conditions on the ABI Prism® 310 Genetic Analyzer (PE Applied Biosystems, Foster City, California).

The California Department of Justice DNA Laboratory currently uses the GS-500-size standard without the 250-base standard assigned and the Local Southern method to size AmpFlSTR® Profiler Plus™ alleles. However, even with the manufacturer's recommended instrument running conditions, studies in this laboratory demonstrate that ambient temperature variation over the course of a 310 run can result in anomalous migration of GS-500 standard fragments. When ambient temperature varies, a simple analysis method change can improve precision.

This study suggests that the Global Southern method may provide improved precision over the Local Southern method when using the GS-500 internal standard with the ABI Prism® 310 Genetic Analyzer. In addition, this study shows that precision for fragments greater than 300 bases is further improved by excluding the 340-base GS-500 fragment in conjunction with using the Global Southern method. When ambient temperature shifts occur, this sizing method change should reduce the number of sample reruns necessary.

Klein et al. (2003) Forensic Sci. Comm. 5(1) Addressing Ambient Temperature Variation Effects on Sizing Precision of AmpFlSTR® Profiler Plus™ Alleles Detected on the ABI Prism® 310 Genetic Analyzer

http://www.fbi.gov/hq/lab/fsc/backissu/jan2003/klein.htm

Thoughts on Size Standards• Be consistent in use if you want to be able to compare

data over time

• All size standards I have tested work

• Allele sizes are different with different internal sizing standards

• GS500 has a large “hole” in its sizing ability when using the local Southern algorithm for medium-sized STR alleles because of the 250 bp peak that cannot be used; also must be run out to 450 bp to be able to type large FGA alleles with ABI kits



Create New Project (Load sample files)Establish Matrix Define Size Standard PeaksSet Analysis Parameters

Minimum peak height (e.g., 50 RFU)Scan range evaluated (best to avoid primer region)Sizing algorithm options (Local Southern is default)

Data Review WindowsAnalysis Control WindowResults Control Window

Steps for Processing Samples:Install matrix onto each (highlighted) sampleSelect appropriate size standard (or create a new one)Check analysis parameter values (or select pre-defined analysis parameter file)Click “Analyze” button

Examine data in Analysis Control by double clicking on sample file name (this allows you to pull up files in all colors and select or de-select color of interest)

Summary of Steps in GeneScan Data Analysis

Confirm that all allele peaks are called in the allelic ladder

Failure to have these alleles designated will result in Genotyper macro failure and inability to type samples

Peak threshold too high so that some alleles are not designated as peaks by the software

Effect of Peak Detection Threshold Genotyper Software

• Converts GeneScan sized peaks into genotype calls using macros

• Genotyping performed by comparison of allele sizes in allelic ladder to sample alleles

Macintosh

2.0

2.5

2.5.2 (5-dye)

Windows NT

3.7 (5-dye)

ABI manual is P/N 904648

Summary of Genotyper Data Analysis

Open template file containing typing macro specific for STR kit used

Import PROCESSED/REVIEWED GeneScan files

Check internal size standard peaks

Run Kazaam (or Power) macro

Review allelic ladder to confirm that all alleles are called correctly

Review sample data by dye color

Remove calls to stutter peaks or instrument artifacts

Create allele table

Export allele table

Rapid Genotyper Data Review

An overlay of electropherograms permits a rapid assessment of sizing precision and the number of alleles seen in a particular sample set…

PowerPlex 16 data viewed in Genotyper 3.7 software

TH01 alleles



Dye blob

STR alleles

stutter

Pull-up (bleed-through)

spike

Blue channel

Green channel

Yellow channel

Red channel

Butler, J.M. (2005) Forensic DNA Typing, 2nd Edition, Figure 15.4, © Elsevier Science/Academic Press

Deciphering Artifacts from the True Alleles

D3S1358

Stutter products

6.0% 7.8%

Incomplete adenylation

D8S1179

-A

+A

-A

+A

Biological (PCR) artifacts

Dye Blobs (“Artifacts”)

DYS437HEX dye blob

Poor primer purity

• Free dye (not coupled to primer) can be injected into the CE capillary and interfere with detection of true STR alleles

• Dye blobs are wider and usually of less intensitythan true STR alleles (amount depends on the purity of the primers used)

• Dye blobs usually appear at an apparent size that is unique for each dye (e.g., FAM ~120 bp, PET ~100 bp)

Butler, J.M., Shen, Y., McCord, B.R. (2003) The development of reduced size STR amplicons as tools for analysis of degraded DNA. J. Forensic Sci 48(5) 1054-1064.

Filtered with Edge columns

Filtered with Edge columns

No Filtering (Straight from PCR)TH01

TPOXCSF1PO

D21S11

D7S820

FGA

TH01

TPOXCSF1PO

D21S11

D7S820

FGA

EDGE GEL FILTRATION CARTRIDGES

Removal of Dye Artifacts Following PCR Amplification

439 389II438

437391 389I

426YCAII

a/b390 385 a/b

393

392H4460

19388

448447

6FAM(blue)

VIC(green)

NED(Yellow)

PET(Red)

LIZ(Orange)

439 389II438437

391 389I

426390

385 a/b

393

392H4460 19

388

448447

100 bp139

200 250* 300150160

340350

YCAII a/b

Residual dye artifacts

100 bp139

200 250* 300150160

340350

Butler, J.M. (2005) Constructing STR multiplex assays. Methods in Molecular Biology: Forensic DNA Typing Protocols(Carracedo, A., ed.), Humana Press: Totowa, New Jersey, in press.

NIST Y-STR 20plex assay

Dye blob removal with Edge columns

Common Errors in Genotyping

• Genotyper table does not import the third allele in a tri-allelic pattern

• Bleedthrough (pull-up) between dye colors results in a peak that falls into a possible allele bin in an adjacent color

• Clicking off a peak label for a true allele by accident and failing to restore the label

• Accidentally clicking on a peak and inserting a label near the beginning of a locus size range that is not a true allele, whichcauses the second true allele to not show up in the final table of results since only two alleles are imported for each locus

GeneMapperIDv 3.1 Software

Since June 2004, Applied Biosystems no longer sells GeneScan and Genotyper software (they will support these programs until June 2009)…ABI is encouraging the adoption of GeneMapperID which replaces the functions of GeneScan and Genotyper.

Released in Nov 2003



310 Data Collection Software and GeneMapperID

• Controls 310 run conditions• Translates light on CCD camera into

electropherogram (raw data)• Sample sheets and injection lists are created

Macintosh

1.0.2

1.2.2

2.1 (5-dye)

Windows NT

1.0 (5-dye)

2.0 (5-dye)

ABI manual is P/N 904958B

Only compatible with GeneMapper software (not back

compatible with GeneScan/Genotyper)

Use of GeneMapperID v3.1

• Projects• Kit, panel, marker, bin• Provides a flexible format and different

methods for viewing data• Can analyze results from multiple STR kits

within the same GeneMapper project file• Not designed to work with pentanucleotide

repeat loci (version 3.2 will be able)

The Project Window Analysis Parameters

Window

Algorithms:

Basic is the default for many applications

Advanced is similar to the Windows NT version of GeneScan

Classic is similar to Macintosh version of GeneScan

Sample Plot Window

GeneScan-like features

Genotyper-like features



Review Multiple Loci from a Single Sample Review Multiple Samples at a Single Locus

Panel Manager Window

Replaces the need to create Genotyper macros

A kit is a collection of panels.A panel is a collection of markers.Bin sets are collections of the expected allele locations for markers contained with a kit.

Process Component Based Quality Values (PQVs)

Green squares = passYellow triangles = checkRed octagons = low quality data

ADO = Allele Display OverflowOS = OffscaleAE = Allele EditLPH = Low Peak HeightSPU = Spectral Pull-UpAN = Allele NumberBD = Broad PeakOVL = OverlapGQ = Genotype Quality

Expert Systems Under Development

• Difference between an “expert system” and a “quality assurance” tool

Examples• TrueAllele (Cybergenetics, Pittsburg, PA)

– Kadash, K. et al. (2004) J. Forensic Sci. 49(4):660-667• STRess2 (Forensic Science Service, UK)• OSIRIS (NCBI/NIJ, Steve Sherry’s team)

• CompareCalls (Myriad Genetics, Salt Lake City, UT)– Ryan, J.H. et al. (2004) J. Forensic Sci. 49(3):492-499

Model for Future Use of Expert Systems

NDIS Board Appendix B

Review of STR profile data



TrueAllele

• TrueAllele® is designed to operate independently of other allele calling systems• A single reviewer with TrueAllele may be used in place of the current two-person

review process• Examined a dataset of 2,048 convicted offender STR profiles and found only

four samples with differences between TrueAllele and Genotyper at a single locus each (spikes, calling alleles outside of ladder range, and Genotyper missing the third allele in a tri-allelic pattern)

Journal of Forensic Sciences (July 2004), volume 49(2), pp. 660-667 Presenting Information Collected on the ABI 310

Steps in Creating PowerPoint Files from GeneScan and Genotyper Data

Steps for Putting STR Data into PowerPoint Presentations

• Open GeneScan or Genotyper file and put data into desired display format– It is better to keep image a medium size that can be

stretched within PowerPoint to make the lines thicker• Take a picture of the desired image on screen

– Windows: press “print screen”, paste image into Paint program, then crop portion of image desired

– Mac: shift-Apple-4 keys, draw box around desired image, open picture image under Macintosh hard drive

• Open PowerPoint and paste copied image from Paint• Label image within PowerPoint• Print as 2-per-page handout to have a nice size

annotated figure

Example with Profiler Plus Allelic Ladder Label Loci…

D3S1358 VWA FGA

Amelogenin

D8S1179 D21S11 D18S51

D5S818 D13S317 D7S820

GS500 ROX internal size standard

Image is too narrow for a nice balanced plot

Stretch the imageResults in thicker lines and more easily read numbers



The same thing is done in Genotyper—display image in software, print screen, crop within Paint and paste into PowerPoint, stretch to fill slide, add labels as needed…

Example Identifiler Data Databank vs Casework Data Challenges

• Databank (single source samples)– Too much DNA may be added to the PCR reaction resulting

in pull-up between dye colors– Lots of data to review – often produced by contractors

• Casework (mixtures or low level samples)– Often limited DNA material to work with– Low copy number samples can result in allele dropout– Can produce complicated STR profiles to interpret

Common Casework Challenges

D3S1358 TH01 D13S317 D16S539 D2S1338

MIXTURES

DEGRADED DNA

D5S818D13S317

D7S820

D16S539 CSF1PO Penta D

From Butler, J.M. (2004) Short tandem repeat analysis for human identity testing. Current Protocols in Human Genetics, John Wiley & Sons, Hoboken, NJ, Unit 14.8, (Supplement 41), pp. 14.8.1-14.8.22

Loss of signal at larger size loci

More than two alleles at multiple loci

suspect

Crime scene evidence(MIXTURE of victim and perpetrator)

All of the suspect’s alleles are included in the evidentiary DNA profile

Forensic Casework DNA Profiles

Profiler Plus STR kit results

Documents

Capillary Electrophoresis in DNA Analysis · in DNA Analysis NEAFS Workshop Mystic, CT September 29-30, 2004 Dr. John M. Butler ... • Review and Test Mixture of dye-labeled PCR